CHROMOGENIC SUBSTANCES IN URINE. 627 



(a) Indoxyl (indigo-blue and indigo-red). Indoxyl (cf. pp. 607 

 and 631) easily oxidises to indigo-blue, or to the isomeric substance 

 indigo-red. The relation between indoxyl and its blue derivative is 

 expressed by the following equation : 



CH + * = C 



(indoxyl) (indigo-blue) 



The formula of indigo-red is C 6 H c = G u N > and it 



arises, like its blue isomer, when, by oxidation, four atoms of hydrogen 

 are removed from two molecules of indoxyl. Oxidising reagents when 

 added to urine may, according to the conditions of the experiment, give 

 rise to the formation of either or both of these coloured derivatives. 

 The blue substance, however, is more easily and more generally 

 obtained. 



It is of great rarity for the urine to be actually pigmented 

 with indigo-blue. As we have already seen (p. 607), the urinary 

 indoxyl is excreted in the form of a conjugated sulphate, and 

 this compound resists oxidation. Only when the indoxyl is first 

 liberated from its combination does the action of oxidising reagents 

 produce the blue colour. It is stated, however, that the urine of 

 cholera may sometimes exhibit a blue shade from the presence of 

 indigo-blue. We have seen that the amount of indoxyl sulphate is 

 increased in the urine whenever bacterial putrefaction of albuminous 

 substances is occurring to a greater extent than usual, whether in the 

 bowel or elsewhere in the body (putrid abscesses, etc.). The most 

 ready method of demonstrating the amount of indoxyl is by converting 

 it into indigo-blue. Jaffa's test. 1 The urine is mixed with an equal 

 bulk of strong hydrochloric acid, by which means the "indican" 

 (indoxyl sulphate) is decomposed and the indoxyl liberated. With a 

 pipette, a solution of a hypochlorite is now added to the mixture drop 

 by drop, when, by oxidation of the indoxyl, indigo-blue is formed. By 

 shaking up the liquid with chloroform, a solution of the blue substance 

 is obtained in the latter (Stockvis, Senator, and others). Otherwise, 

 a crystal of potassium chlorate is placed at the bottom of a test tube 

 and covered with the urine to be examined. Strong hydrochloric acid 

 is then allowed to run down the side of the tube so as to reach the 

 crystal without mixing with the urine. The latter floats upon the acid, 

 and at the junction of the fluids a blue ring is seen of intensity varying 

 with the amount of indoxyl present. 



But indigo-blue is itself an easily oxidisable substance. It is 

 instantly decolorised by nitric acid, and without difficulty by hypo- 

 chlorites. In Jaffe's test, as above described, it is therefore necessary 

 to add the oxidising agent with great care, or the blue colour will 

 disappear as soon as formed. In Obermayer's 2 method, the urine is 

 first precipitated by acetate of lead, and filtered ; to the filtrate is added 

 an equal bulk of strong hydrochloric acid, containing two or three parts 

 per thousand of ferric chloride. The mixture is shaken for a short 

 time, and the liberated pigment taken up, as before, in chloroform. In 



1 Arch.f. d. ges. PhysioL, Bonn, 1870, Bd. iii. S. 448. 



2 Wien. Tclin. Wchnschr., 1890, S. 176. 



